110
M. Scarsini et al.
5.5 Genetic Engineering
The amount of carotenoid produced by microalgae can be also enhanced using genetic
engineering. Theoretically, these methods allow to combine the quality aspects of
carotenoid production i.e. the type of carotenoid and the quantity through cloning in
adequate producing organisms (e.g. bacteria: Takemura et al. (2019), fungus: Misawa
and Shimada (1998), Miura et al. (1998), microalga: Leon et al. (2007)). First trials to
produce carotenoids using genetic engineering consisted in expressing genes various
types of organisms. For instance, the β-carotene ketolase and β-carotene oxidase from
Haematococcus pluvialis were expressed in a strain of Escherichia coli producing
β-carotene and zeaxanthin (Harker and Hirschberg 1997), the cyanobacterium Synechococcus PCC7942 (Breitenbach et al. 1996) and the land plant Arabidopis (Zhong
et al. 2011). In Chlamydomonas reinhardtii, the expression of the Haematococcus
pluvialis bkt gene led to transgenic strains able to synthesize keto-carotenoids not
present in the wild strain (Leon et al. 2007). The overexpression of a mutant version
of the pds gene in Chromochloris zofingiensis and Haematococcus pluvialis led to
increased levels of total carotenoids (+32% for Chlorella) and astaxanthin (+54% for
Chlorella and +26% for Haematococcus) in transformants (Liu et al. 2013a, Steinbrenner and Sandmann 2006). Expression of the endogenous nuclear pds gene in
the chloroplast of Haematococcus pluvialis showed up higher astaxanthin accumulation (Galarza et al. 2018). Chlamydomonas strains overexpressing exogenous psy
genes (from Dunaliella salina and Chromochloris zofigiensis) were able to display
a 2 fold increase in lutein level (Couso et al. 2011; Cordero et al. 2011). However,
not all microalgal cloned genes are active in bacteria. For instance, PtZEP1, coding
zeaxanthin epoxidase in the diatom Phaeodactylum tricornutum does not show any
activity in Escherichia coli (Eilers et al. 2016). The homologous expression of
cyanobacterial genes was also tested. For instance, Lagarde et al. (2000) reported the
increase of zeaxanthin and other carotenoid production in engineered Synechocystis
sp PCC6803. Recently, Ye and Huang (2019) reported the selection of mutants of
Chromochloris zofingiensis ATCC 30412 using a random mutagenesis strategy. The
mutants accumulate only traces of canthaxanthin and astaxanthin unless they are
grown mixotrophically on glucose as an alternative carbon source. In these conditions, the content of astaxanthin was reduced when compared to the wild-type but
the amount of zeaxanthin was 7–11 higher.
5.6 Conclusion and Perspectives
The production of carotenoids by microalgae requires an optimized growth medium
that might be taxon specific and biotechnological processes for. For instance, the
production of astaxanthin by Haematococcus pluvialis is a two-step process because
the stresses triggering astaxanthin accumulation inhibit cell division. The biomass
M. Scarsini et al.
5.5 Genetic Engineering
The amount of carotenoid produced by microalgae can be also enhanced using genetic
engineering. Theoretically, these methods allow to combine the quality aspects of
carotenoid production i.e. the type of carotenoid and the quantity through cloning in
adequate producing organisms (e.g. bacteria: Takemura et al. (2019), fungus: Misawa
and Shimada (1998), Miura et al. (1998), microalga: Leon et al. (2007)). First trials to
produce carotenoids using genetic engineering consisted in expressing genes various
types of organisms. For instance, the β-carotene ketolase and β-carotene oxidase from
Haematococcus pluvialis were expressed in a strain of Escherichia coli producing
β-carotene and zeaxanthin (Harker and Hirschberg 1997), the cyanobacterium Synechococcus PCC7942 (Breitenbach et al. 1996) and the land plant Arabidopis (Zhong
et al. 2011). In Chlamydomonas reinhardtii, the expression of the Haematococcus
pluvialis bkt gene led to transgenic strains able to synthesize keto-carotenoids not
present in the wild strain (Leon et al. 2007). The overexpression of a mutant version
of the pds gene in Chromochloris zofingiensis and Haematococcus pluvialis led to
increased levels of total carotenoids (+32% for Chlorella) and astaxanthin (+54% for
Chlorella and +26% for Haematococcus) in transformants (Liu et al. 2013a, Steinbrenner and Sandmann 2006). Expression of the endogenous nuclear pds gene in
the chloroplast of Haematococcus pluvialis showed up higher astaxanthin accumulation (Galarza et al. 2018). Chlamydomonas strains overexpressing exogenous psy
genes (from Dunaliella salina and Chromochloris zofigiensis) were able to display
a 2 fold increase in lutein level (Couso et al. 2011; Cordero et al. 2011). However,
not all microalgal cloned genes are active in bacteria. For instance, PtZEP1, coding
zeaxanthin epoxidase in the diatom Phaeodactylum tricornutum does not show any
activity in Escherichia coli (Eilers et al. 2016). The homologous expression of
cyanobacterial genes was also tested. For instance, Lagarde et al. (2000) reported the
increase of zeaxanthin and other carotenoid production in engineered Synechocystis
sp PCC6803. Recently, Ye and Huang (2019) reported the selection of mutants of
Chromochloris zofingiensis ATCC 30412 using a random mutagenesis strategy. The
mutants accumulate only traces of canthaxanthin and astaxanthin unless they are
grown mixotrophically on glucose as an alternative carbon source. In these conditions, the content of astaxanthin was reduced when compared to the wild-type but
the amount of zeaxanthin was 7–11 higher.
5.6 Conclusion and Perspectives
The production of carotenoids by microalgae requires an optimized growth medium
that might be taxon specific and biotechnological processes for. For instance, the
production of astaxanthin by Haematococcus pluvialis is a two-step process because
the stresses triggering astaxanthin accumulation inhibit cell division. The biomass